Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Swarm rat chondrosarcoma cell cultures were metabolically labeled with [35S]sulfate, [3H]glucose, or [3H]glucosamine. Chondroitin sulfate chains were isolated from purified aggrecan using alkaline borohydride treatment and Superose 6 chromatography. Various linkage region oligosaccharide alditols were derived from these chains using sequential chondroitinase digestions (ABC lyase followed by ACII lyase). They were then further processed by mercuric acetate treatment, which removed the 4,5-unsaturated uronosyl residue from the nonreducing end of the linkage, and then beta-galactosidase digestion which liberated the 2 galactose residues from the xylitol reducing terminus. Alkaline phosphatase digestions were performed to verify the presence of phosphate esters. All linkage region structures were isolated and identified using a combination of Progel-TSK G2500 and CarboPac PA1 chromatography steps in conjunction with monosaccharide analyses. This study revealed that chondroitin sulfate chains from aggrecan synthesized by rat chondrosarcoma cells in vitro have the following properties: 1) three out of every four of their linkage regions carry a phosphate ester on xylose, 2) nearly three out of every five chains begin the repeating disaccharide region with an unsulfated first disaccharide unit, 3) nearly twice as many nonphosphorylated chains have a sulfated first disaccharide than their phosphorylated counterparts, and 4) the vast majority of these chains do not contain sulfated galactose in their linkage regions. This report also describes a borohydride reduction procedure to confer alkali stability to the 3-substituted, unsaturated disaccharides derived from chondroitinase digests of chondroitin sulfate. Furthermore, a CarboPac PA1 method is demonstrated that separates these reduced disaccharides with exceptional resolution.
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PMID:Structural analysis of the linkage region oligosaccharides and unsaturated disaccharides from chondroitin sulfate using CarboPac PA1. 155 66

We characterized catabolite repression of the genes encoding xylose utilization in Bacillus megaterium. A transcriptional fusion of xylA encoding xylose isomerase to the spoVG-lacZ indicator gene on a plasmid with a temperature-sensitive origin of replication was constructed and efficiently used for single-copy replacement cloning in the B. megaterium chromosome starting from a single transformant. In the resulting strain, beta-galactosidase expression is 150-fold inducible by xylose and 14-fold repressed by glucose, showing that both regulatory effects occur at the level of transcription. Insertion of a kanamycin resistance gene into xylR encoding the xylose-dependent repressor leads to the loss of xylose-dependent regulation and to a small drop in the efficiency of glucose repression to eightfold. Deletion of 184 bp from the 5' part of the xylA reading frame reduces glucose repression to only twofold. A potential glucose-responsive element in this region is discussed on the basis of sequence similarities to other glucose-repressed genes in Bacillus subtilis. The sequence including the glucose-responsive element is also necessary for repression exerted by the carbon sources fructose and mannitol. Their efficiencies of repression correlate to the growth rate of B. megaterium, as is typical for catabolite repression. Glycerol, ribose, and arabinose exert only a basal twofold repression of the xyl operon, which is independent of the presence of the cis-active glucose-responsive element within the xylA reading frame.
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PMID:Catabolite repression of the xyl operon in Bacillus megaterium. 156 31

Induction of the lactose-galactose regulon is strongly repressed by glucose in some but not all strains of Kluyveromyces lactis. We show here that in strongly repressed strains, two to three times less Kl-GAL4 mRNA is synthesized and that expression of structural genes in the regulon such as LAC4, the structural gene for beta-galactosidase, is down regulated 40-fold or more. Comparative analysis of strains having a strong or weak repression phenotype revealed a two-base difference in the promoter of the Kl-GAL4 (also called LAC9) positive regulatory gene. This two-base difference is responsible for the strong versus the weak repression phenotype. The two base changes are symmetrically located in a DNA sequence having partial twofold rotational symmetry (14 of 21 bases). We hypothesize that this region functions as a sensitive regulatory switch, an upstream repressor sequence (URS). According to our model, the presence of glucose in the culture medium signals, by an unidentified pathway, a repressor protein to bind the URS. Binding reduces transcription of the Kl-GAL4 gene so that the concentration of the Kl-GAL4 protein falls below the level needed for induction of LAC4 and other genes in the regulon. For strains showing weak glucose repression, we hypothesize that the two base changes in the URS reduce repressor binding so that the regulon is not repressed. Our results illustrate an important principle of genetic regulation: a small (2- to 3-fold) change in the concentration of a regulatory protein can produce a large (40-fold or greater) change in expression of structural genes. This mechanism of signal amplification could play a role in many biological phenomena that require regulated transcription.
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PMID:The signal for glucose repression of the lactose-galactose regulon is amplified through subtle modulation of transcription of the Kluyveromyces lactis Kl-GAL4 activator gene. 156 29

The RAG1 gene encodes a membrane protein involved in the low-affinity glucose/fructose transport system of the yeast Kluyveromyces lactis. Analysis of steady-state mRNA levels analysis and quantitation of expression by beta-galactosidase from RAG1-lacZ fusions assays revealed that the RAG1 gene was poorly expressed in cells grown under gluconeogenesis conditions, but was induced more than ten-fold when they were grown on various sugars. These sugars included glucose, fructose, mannose, sucrose, raffinose, as well as galactose. Nucleotide sequence and deletion analysis of the 5' flanking region of the RAG1 gene showed that an essential cis-acting element required for induced transcription of the RAG1 gene resided between -615 and -750 from the coding sequence. This region contained a 22 bp purine stretch, and a pair of 11 bp direct repeat sequences. The 11 bp repeats harbor a CCAAT motif, a consensus sequence for binding of the yeast and mammalian HAP2/3/4-type protein complex. The transcription of the RAG1 gene was dramatically affected by three unlinked mutations, rag4, rag5 and rag8. We discuss the possible roles of RAG4, RAG5 and RAG8 gene products in the expression of the RAG1 gene, as well as the importance of the inducible RAG1 gene in the fermentative growth of K. lactis.
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PMID:Glucose transport in the yeast Kluyveromyces lactis. II. Transcriptional regulation of the glucose transporter gene RAG1. 160 79

We have examined the expression of the gene encoding the iron-protein subunit (Ip) of succinate dehydrogenase in Saccharomyces cerevisiae. The gene had been cloned by us and shown to be subject to glucose regulation (A. Lombardo, K. Carine, and I. E. Scheffler, J. Biol. Chem. 265:10419-10423, 1990). We discovered that a significant part of the regulation of the Ip mRNA levels by glucose involves the regulation of the turnover rate of this mRNA. In the presence of glucose, the half-life appears to be less than 5 min, while in glycerol medium, the half-life is greater than 60 min. The gene is also regulated transcriptionally by glucose. The upstream promoter sequence appeared to have four regulatory elements with consensus sequences shown to be responsible for the interaction with the HAP2/3/4 regulatory complex. A deletion analysis has shown that the two distal elements are redundant. These measurements were carried out by Northern (RNA) analyses of Ip mRNA transcripts as well as by assays of beta-galactosidase activity in cells carrying constructs of the Ip promoter linked to the lacZ coding sequence. These observations on the regulation of mRNA stability were also extended to the mRNA of the flavoprotein subunit of succinate dehydrogenase and in some experiments of iso-1-cytochrome c.
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PMID:Control of mRNA turnover as a mechanism of glucose repression in Saccharomyces cerevisiae. 162 Jan 7

The genes coding for the binding-protein-dependent lactose transport system and beta-galactosidase in Agrobacterium radiobacter strain AR50 were cloned and partially sequenced. A novel lac operon was identified which contains genes coding for a lactose-binding protein (lacE), two integral membrane proteins (lacF and lacG), an ATP-binding protein (lacK) and beta-galactosidase (lacZ). The operon is transcribed in the order lacEFGZK. The operon is controlled by an upstream regulatory region containing putative -35 and -10 promoter sites, an operator site, a CRP-binding site probably mediating catabolite repression by glucose and galactose, and a regulatory gene (lacl) encoding a repressor protein which mediates induction by lactose and other galactosides in wild-type A. radiobacter (but not in strain AR50, thus allowing constitutive expression of the lac operon). The derived amino acid sequences of the gene products indicate marked similarities with other binding-protein-dependent transport systems in bacteria.
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PMID:Molecular analysis of the lac operon encoding the binding-protein-dependent lactose transport system and beta-galactosidase in Agrobacterium radiobacter. 163 Mar 15

The effect of cyclic 3',5'-adenosine monophosphate (cAMP) on the rate of beta-galactosidase biosynthesis was studied in the cells of Escherichia coli M-17 growing in MPB and mineral media with glucose and maltose, i.e. under the conditions of various catabolite repression, as well as upon lac-operon induction by isopropyl-beta-D-galactopyranoside (IPGP). The stimulating action of exogenous cAMP was found only in a medium with salts and glucose. The induction by IPGP was highest during the growth in a medium with glucose and maltose. When the medium contained IPGP, cAMP accelerated the enzyme synthesis in all media, but only at the early growth phases, while cAMP eliminated the effect of IPGP at the stationary phase of growth. The regulation of beta-galactosidase biosynthesis by cAMP demonstrated for the first time that this effect depended on the physiological state of E. coli: the expression of catabolite-sensitive E. coli genes was subject to both positive and negative regulation in one and the same inducible system. The effect exerted by cAMP depended on the nature of a carbon source in the growth medium.
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PMID:[Regulation of beta-galactosidase synthesis in Escherichia coli by exogenous cyclic 3',5'-adenosine monophosphate]. 165 99

Lactobacillus plantarum ATCC 8014 grew poorly on raffinose agar plates, but large mutant colonies appeared in high frequency from a thin film of background growth. The alpha-galactosidase and beta-galactosidase activities of L. plantarum ATCC 8014 and a mutant strain were studied in static cultures and pH-controlled fermenter cultures. Both alpha-galactosidase and beta-galactosidase production were inducible in the parental strain; the induction was not needed in the mutant. The alpha-galactosidase activity of both strains was repressed by glucose but not by alpha-methyl-D-glucoside. The mutant phenomenon might be an obstacle in connection to traditional Lactobacillus identification by means of carbohydrate fermentation.
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PMID:Spontaneous mutations changing the raffinose metabolism of Lactobacillus plantarum. 166 2

The complete nucleotide sequences of the genes encoding aldose 1-epimerase (mutarotase) (galM) and UDPglucose 4-epimerase (galE) and flanking regions of Streptococcus thermophilus have been determined. Both genes are located immediately upstream of the S. thermophilus lac operon. To facilitate the isolation of galE, a special polymerase chain reaction-based technique was used to amplify the region upstream of galM prior to cloning. The galM protein was homologous to the mutarotase of Acinetobacter calcoaceticus, whereas the galE protein was homologous to UDPglucose 4-epimerase of Escherichia coli and Streptomyces lividans. The amino acid sequences of galM and galE proteins also showed significant similarity with the carboxy-terminal and amino-terminal domains, respectively, of UDPglucose 4-epimerase from Kluyveromyces lactis and Saccharomyces cerevisiae, suggesting that the yeast enzymes contain an additional, yet unidentified (mutarotase) activity. In accordance with the open reading frames of the structural genes, galM and galE were expressed as polypeptides with apparent molecular masses of 39 and 37 kilodaltons, respectively. Significant activities of mutarotase and UDPglucose 4-epimerase were detected in lysates of E. coli cells containing plasmids encoding galM and galE. Expression of galE in E. coli was increased 300-fold when the gene was placed downstream of the tac promoter. The gene order for the gal-lac gene cluster of S. thermophilus is galE-galM-lacS-lacZ. The flanking regions of these genes were searched for consensus promoter sequences and further characterized by primer extension analysis. Analysis of mRNA levels for the gal and lac genes in S. thermophilus showed a strong reduction upon growth in medium containing glucose instead of lactose. The activities of the lac (lactose transport and beta-galactosidase) and gal (UDPglucose 4-epimerase) proteins of lactose- and glucose-grown S. thermophilus cells matched the mRNA levels.
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PMID:Carbohydrate utilization in Streptococcus thermophilus: characterization of the genes for aldose 1-epimerase (mutarotase) and UDPglucose 4-epimerase. 169 27

We inserted a full-length murine cDNA, which had been isolated from F9 embryonal carcinoma cells by using a bovine lactose synthetase A protein cDNA as a probe, in a mammalian expression vector (pCMGT1) and expressed it in COS-1 cells to characterize the pCMGT1-directed enzyme. The galactosyltransferase activity toward asialo-agalacto-transferrin (AsAg-Tf) in the pCMGT1-transfected cells was approximately eightfold higher than that in mock- or non-transfected cells. In contrast, no difference was observed in the specific activity of galactose transfer between pCMGT1-transfected cells and mock- or non-transfected cells when asialo-ovine submaxillary mucin were used as an acceptor. Since almost all [3H]galactose incorporated into the AsAg-Tf was released by digestion with streptococcal beta-galactosidase, most of the linkage created by this enzyme was in the Gal beta 1-4GlcNAc group. The acceptor specificity of the pCMGT1-directed enzyme was changed from N-acetylglucosamine to glucose by adding alpha-lactalbumin in the reaction mixture. Alpha-Lactalbumin also partially inhibited the galactose transfer to AsAg-Tf. The kinetic study revealed that the apparent Km values of the pCMGT1-directed enzyme for N-acetylglucosamine, AsAg-Tf and UDP-Gal are 2 mM, 60 microM and 24 microM, respectively. These results indicated that the murine cDNA isolated from F9 cells encodes an active enzyme which catalyzes not only the lactose synthesis but also the transfer of galactose to N-acetylglucosamine residues of Asn-linked sugar chains of glycoproteins in a beta 1-4 linkage.
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PMID:Characterization of a murine beta 1-4 galactosyltransferase expressed in COS-1 cells. 170 63


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